The present invention relates to supports used in DNA and RNA synthesis. The supports according to the invention do not include a pre-attached nucleoside, and therefore can be used universally for any DNA or RNA synthesis, irrespective of the desired terminal nucleoside. Moreover, the supports according to the invention allow fast cleavage and dephosphorylation at relatively low temperatures and under relatively mild reaction conditions.
A vast majority of oligonucleotide syntheses are carried out on supports to which a first nucleoside has been pre-attached (e.g., by a succinate or hydroquinone-O,Oxe2x80x2-diacetate linker). After routine cleavage and deprotection steps, this nucleoside becomes the 3xe2x80x2 terminal nucleoside of the target oligonucleotide. For standard DNA and RNA synthesis, this requires an inventory of four deoxynucleoside and four ribonucleoside supports, i.e., a different support depending on the desired terminal nucleoside.
A xe2x80x9cuniversalxe2x80x9d solid support (i.e., a support without the first nucleoside attached) for DNA and RNA synthesis that permitted direct coupling of any residue and elimination of the terminal phosphodiester linkage at the same time as the deprotection step would offer several advantages. For example, such a universal support would: (a) eliminate the need for an inventory of several different nucleoside supports; (b) minimize the possibility of error in the selection of the correct support type; (c) reduce the time and eliminate errors in the generation of an array of nucleoside supports in 96 well synthesizers; and (d) allow the preparation of oligonucleotides containing a 3xe2x80x2-terminal nucleoside which is not available as a support.
In spite of these major potential benefits of universal supports, at the present time, the use of universal supports for DNA and RNA synthesis has not been favored. One major hurdle to overcome relates to finding conditions to eliminate the terminal phosphate, produced from the first nucleoside phosphoramidite addition cycle to the required terminal hydroxyl group. This problem is considered in more detail below.
Certain known universal supports based on the ribonucleoside elimination model are known as xe2x80x9cMcLean supports.xe2x80x9d See S. Scott, P. Hardy, R. C. Sheppard, and M. J. McLean, xe2x80x9cA Universal Support for Oligonucleotide Synthesis,xe2x80x9d Innovation and Perspectives in Solid Phase Synthesis, 3rd International Symposium, 1994, pp. 115-124, which document is entirely incorporated herein by reference. McLean supports also are described in U.S. Pat. Nos. 5,681,945 and 5,886,193, which patents are entirely incorporated herein by reference. A McLean support also is illustrated in FIG. 1a. This solid support allows the detritylation, the addition of the first nucleoside monomer, and the remainder of the oligomer preparation to proceed without any changes from standard protocols. Elimination of the terminal phosphodiester group utilizes the same reagents needed for routine deprotection of oligonucleotides, but requires more aggressive and lengthy treatment conditions (e.g., concentrated ammonium hydroxide/80xc2x0 C./17 hours as compared to the standard oligomer deprotection conditions of ammonium hydroxide/55xc2x0 C./5-6 hours). While these more aggressive conditions are suitable for the preparation of unmodified oligonucleotides, they are not compatible with base-labile nucleoside analogues. Furthermore, prolonged treatment with basic volatile reagents or the need to employ a desalting step in the case of sodium hydroxide make this solid support unattractive for use in industrial multi-well synthesizers.
The instability of RNA to strongly basic conditions is caused by the proximity of the 2xe2x80x2-OH group to the phosphodiester group. Attack of the 2xe2x80x2-OH on the adjacent phosphorus gives rise to an energetically favorable 5-membered transition state, which can open up again to form a mixture of 2xe2x80x2- and 3xe2x80x2-phosphodiester internucleotide linkages, or can lead to chain scission by elimination of the 3xe2x80x2- or 5xe2x80x2-hydroxyl group. In the case of a McLean universal support of FIG. 1a, cleavage from the support by hydrolysis of a succinate or, better, a hydroquinone-O,Oxe2x80x2-diacetate (Q) linkage, generates a hydroxyl group adjacent to the terminal phosphodiester linkage. Additional base treatment leads to the elimination of the terminal phosphate group and formation of the desired 3xe2x80x2-OH. Wengel and coworkers described a similar strategy using a neighboring hydroxyl group to facilitate elimination. See C. Scheuer-Larsen, C. Rosenbohm, T. J. D. Jorgensen, and J. Wengel, xe2x80x9cIntroduction of a Universal Solid Support for Oligonucleotide Synthesis,xe2x80x9d Nucleosides and Nucleotides, 1997, Vol. 16, pp. 67-80, which article is entirely incorporated herein by reference. Lyttle and a group at Biosearch Technologies described a variation of the Wengel procedure. The Lyttle procedure used a linkage to a polymeric support that is not hydrolyzed by base, so that extended base treatment releases the dephosphorylated oligo, while leaving any undesired by-product still attached to the support. See M. H. Lyttle, D. J. Dick, D. Hudson, and R. M. Cook, xe2x80x9cA Phosphate Bound Universal Linker for DNA Synthesis,xe2x80x9d Nucleosides and Nucleotides, 1999, Vol. 18, pp. 1809-1824, which article is entirely incorporated herein by reference.
The main impediment to adoption of a universal support has been the aggressively basic conditions required to complete the elimination reaction to release the terminal hydroxyl group. The standard reagents used in oligonucleotide deprotection are ammonium hydroxide and aqueous methylamine, which are popular because they are readily available and completely volatile. Using these reagents to carry out the elimination reaction, however, requires either high temperature, with attendant high pressure, or extended reaction times. The situation can be improved by adding metal ions to the mix (such as Li+, Na+, and Zn2+), to speed up the elimination reaction, presumably by stabilizing the 5-membered transition state. However, the speed and simplicity of evaporation of the deprotection solution to give the crude oligonucleotide without desalting is not possible when these ionic additives are used.
Other known universal supports that use neighboring aminomethyl or diamino-ethyl groups to assist the elimination reaction have been described by Azhayev. See A. V. Azhayev, xe2x80x9cA New Universal Solid Support for Oligonucleotide Synthesis,xe2x80x9d Tetrahedron, 1999, Vol. 55, pp. 787-800, which article is entirely incorporated herein by reference. These supports, which are illustrated in FIGS. 1b, 1c, and 1d, are not only compatible with the preparation of all common types of oligonucleotides, but they also function well for oligomers with unusual base labile nucleoside units. Using volatile ammonium hydroxide or aqueous methylamine with these supports (e.g., concentrated ammonium hydroxide/80xc2x0 C./2-8 hours), 3xe2x80x2-terminal dephosphorylation was significantly speeded up, and dephosphorylation could be achieved under neutral conditions using aqueous zinc chloride or water. In these supports, the neighboring aminomethyl- or diaminoethyl-groups assist for the elimination of the terminal phosphodiester to generate termini with 3xe2x80x2-hydroxyl.
While the universal supports of FIGS. 1b-1d offer genuine advantages, especially if the oligonucleotides contain base-labile components, they still are not ideal for mainstream applications. These supports still require relatively long treatment times with basic volatile reagents at elevated temperatures. These features make these supports somewhat unattractive for industrial applications.
Accordingly, there is a need in the art for a universal support suitable for DNA and RNA synthesis that eliminates the drawbacks mentioned above. A desirable universal support would allow fast cleavage and dephosphorylation at relatively low temperatures (e.g., in 20 minutes at room temperature), and under relatively mild reaction conditions (e.g., using a 2M ammonia in methanol cleavage reagent). Moreover, the desired universal support would be compatible for use under ultra-mild, normal, and ultra-fast deprotection conditions. Finally, the desired universal support product will be cost-effective (e.g., comparable in price to regular 2xe2x80x2-deoxynucleoside supports).
The present invention relates to universal supports for oligonucleotide, DNA, and RNA synthesis, as well as to methods of making and using the supports. A first aspect of the invention relates to a universal support material represented by the following formula: 
In this formula, substituent A may be H, an alkyl group, an aryl group, a polymeric base material, or a silica base material; substituent B may be an acyl group, an aroyl group, a polymeric base material, or a silica base material; and substituent C may be a dimethoxytrityl group or another protecting group removable under acidic or neutral conditions. For the support material according to this formula, one of substituent A or B constitutes a polymeric base material or a silica base material.
In one embodiment of the invention, substituent A constitutes the polymeric or silica base material. As one example, the silica base material of substituent A may be a long chain alkylamino controlled pore glass base material. As an even more specific example, substituent A may include the following substituent: 
wherein lcaaCPG and its accompanying substituent groups represent a long chain alkylamino controlled pore glass base material. As illustrative examples, in this embodiment of the invention (where substituent A is the base material), substituent B may be an acyl group (e.g., a formyl group or an acetyl group) or an aroyl group (e.g., a phenoxyacetyl group). Also, optionally, the acyl or aroyl groups may be substituted with halogen atoms (e.g., chloroacetyl, dichloroacetyl, 4-chlorophenoxyacetyl, and 2,4-dichlorophenoxyacetyl). Other suitable substituent groups can be used as substituent B without departing from the invention.
In another embodiment of the invention, substituent B may constitute the polymeric or silica base material. As one example, the silica base material of substituent B may be a long chain alkylamino controlled pore glass base material. As an even more specific example, substituent B may include the following substituent group: 
wherein lcaaCPG and its accompanying substituent groups represent the long chain alkylamino controlled pore glass base material. In this embodiment, substituent A may be various suitable substituent groups. Examples of these suitable substituent groups include: hydrogen, an alkyl group and a phenyl group, and optionally these groups can be substituted, e.g., with halogen atoms. More specific examples of suitable substituent groups useful as substituent A include: a xe2x80x94CF3 group, a methyl group, an ethyl group, a propyl group, a butyl group, and a t-butyl group.
A second aspect of the present invention relates to articles of manufacture represented by the following formula: 
wherein substituent A may be H, an alkyl group, an aryl group, a polymeric base material, or a silica base material; substituent B may be an acyl group, an aroyl group, a polymeric base material, or a silica base material; and substituent C may be a dimethoxytrityl group, a nucleotide-containing group, or a protecting group removable under acidic or neutral conditions. Again, in this aspect of the invention, one of substituents A or B constitutes a polymeric base material or a silica base material. In this aspect of the invention, substituents A and B may constitute the various groups described above with respect to the first aspect of the invention.
In these articles of manufacture according to the invention, substituent C may be a nucleotide-containing group. In other words, this aspect of the invention also relates to the solid supports according to the invention with one or more nucleotides supported thereon. For example, the nucleotide-containing group supported on the universal support may include at least one nucleotide selected from the group consisting of: thymidine, 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxycytindine, and 2xe2x80x2-deoxyguanosine. The nucleotide-containing group may be attached to the support via a phosphotriester group (at substituent C).
Various specific support materials according to the invention include the following: 
In these illustrated formulae, xe2x80x9cDMTrxe2x80x9d represents a dimethoxytrityl group, and xe2x80x9clcaaCPGxe2x80x9d and its accompanying substituent groups represent a long chain alkylamino controlled pore glass base material. This invention also relates to the resulting articles of manufacture produced when one or more nucleotides are supported on the above support materials, e.g., via a phosphotriester linkage at the DMTr group.
Another aspect of this invention relates to an article of manufacture represented by the following formula: 
wherein X represents xe2x95x90O, S, or NH; Y represents O or NH; substituent A represents H, an alkyl group, an aryl group, a polymeric base material, a silica base material, a substituent including an activated ester capable of coupling to an amino support, or a substituent terminating in a phosphoramidite group capable of coupling to a support or an oligonucleotide; substituent B represents a first protecting group; and substituent C represents a second protecting group that is different from the first protecting group. In this aspect of the invention, one of the first protecting group or the second protecting group is a substituent group capable of being removed under neutral conditions (e.g., a base-labile protecting group, such as dichloroacetyl or other acyl or aroyl groups), and the other of the first protecting group or the second protecting group is a substituent group capable of being removed under acidic or specific neutral conditions. For example, in one embodiment of this aspect of the invention, substituent B may be a base-labile group, such as an acyl or an aroyl group, and substituent C may be a dimethoxytrityl group. In another embodiment of this aspect of the invention, substituent C may be a base-labile group, such as an acyl group or an aroyl group, and substituent B may be a dimethoxytrityl group.
Another aspect of this invention relates to use of the support products. In one embodiment, this invention relates to a method for releasing a product oligonucleotide from a support by treating a supported oligonucleotide with a nonaqueous base (which may be an anhydrous base). In this embodiment of the invention, the supported oligonucleotide is represented by the following formula: 
wherein substituent A includes H, an alkyl group, an aryl group, or a polymeric or silica support material, substituent B is an acyl group, an aroyl group, or a polymeric or silica support, and substituent C is an oligonucleotide-containing group, with the proviso that one of substituents A or B constitutes a polymeric or silica support material. This nonaqueous base treatment acts to cleave substituent A or B (whichever is not the polymeric or silica support) from the supported oligonucleotide. Thereafter, the supported oligonucleotide is further treated to separate the product oligonucleotide (included as part of substituent C) from the support (included as part of substituent A or B). The nonaqueous base used in the initial treatment step may include ammonia gas or ammonia, such as ammonia in an alcohol solution (e.g., in methanol).
Various support materials described above may be used in performing this method. For example, substituent A may include a long chain alkylamino controlled pore glass base material. In this instance, as additional examples, substituent B may be a chloroacetyl group, a dichloroacetyl group, a 4-chlorophenoxyacetyl group, a 2,4-dichlorophenoxyacetyl group, or any of the other substituent groups mentioned above.
Specific examples of the supported oligonucleotide in this embodiment of the invention include the following: 
wherein lcaaCPG and its accompanying substituent groups represent a long chain alkylamino controlled pore glass base material. The oligonucleotide-containing group may be attached via a phosphotriester group.